The microbiota of the human vagina can profoundly affect the health of women and neonates. For instance, women with the condition bacterial vaginosis (BV) have increased risks of acquiring sexually transmitted infections such as HIV, and pregnant women with BV have increased risk of preterm birth. Our understanding of BV is hampered by the failure to cultivate many of the bacteria associated with this condition. PCR methods have demonstrated that novel and uncultivated bacterial species are common in women with BV and some uncultivated bacteria are associated with important adverse outcomes such as antibiotic failure and ascending infection. Microbial genome sequencing efforts hold the promise of providing new insights regarding the metabolic interactions among vaginal bacteria that help sustain these vaginal communities and the pathogenic capabilities of key species that mediate poor health outcomes. Conventional cultivation methods may fail to propagate human-associated bacteria for a variety of reasons. For example, growth in monoculture may be precluded when bacterial species grow together as metabolic (syntrophic) partners, exchanging critical nutrients. Likewise, one species may depend on a second species for a signaling compound that stimulates cell division, again precluding monoculture. Alternatively, conventional laboratory medium may fail to replicate the local environment found in a body surface or cavity. We seek to overcome these limitations using several different novel cultivation technologies and approaches, which are all based on the principle that cultivation of vaginal bacteria can best be achieved by better reproducing the natural vaginal microenvironment. In Aim 1 we will use a novel miniaturized diffusion chamber device to isolate and propagate bacteria in microliter scale chambers. The in situ isolation chip (iChip) will allow bacteria to grow in pure culture while bathed in fluid from the natural environment. In this case, we will use vaginal lavage fluid for in vitro propagation of bacteria, and the human vagina itself for short term in vivo propagation of bacteria. The iChip serves as an intermediate for adapting bacteria for independent growth on laboratory media, a process that we call domestication. In Aim 2, we will use co-cultivation methods to propagate fastidious vaginal bacteria in pure culture. This approach uses two growth chambers that are separated by a membrane permeable to chemicals but not bacteria. A known cultivated bacterium or bacterial community is inoculated into the lower chamber, and an uncultivated bacterial cell is inoculated in the upper chamber. Bacteria growing in the lower chamber are allowed to produce nutrients and growth factors to stimulate the proliferation of fastidious bacteria in the upper chamber. This strategy takes advantage of nutrient cycling and cross species signaling between bacterial species to allow isolation of fastidious microbes. In Aim 3, we will use conventional media supplemented with sterile filtered vaginal lavage fluid to cultivate fastidious vaginal bacteria under diverse conditions with prolonged incubation. This media will contain missing nutrients and signaling molecules from the vaginal environment to facilitate isolation. Some novel vaginal bacteria are capable of laboratory propagation but require extended growth times and very specific growth conditions. Furthermore, some bacteria can be cultivated in the lab but are not easily identified using phenotypic methods. We will use 16S rRNA gene PCR to facilitate identification of all bacteria in this study. PUBLIC HEALTH RELEVANCE: The vaginal microbiota has a major impact on the health of women and neonates. Bacterial vaginosis affects 29% of women in the United States and is associated with increased risk of sexually transmitted diseases, including HIV, preterm birth, pelvic inflammatory disease, and several other adverse outcomes. The microbiology of bacterial vaginosis is poorly understood and many bacterial species found in this condition have not been cultivated in the laboratory. This application seeks to cultivate numerous fastidious vaginal bacteria associated with bacterial vaginosis using several novel cultivation approaches with the goal of gaining new insights about the vaginal microbiome and its role in human health.